Bottom Line:
Under anesthesia, 6 fetal sheep at 0.85 gestation were instrumented with vascular catheters and a Transonic flow probe around a femoral artery.This technique permits blockade of de novo synthesis of NO while compensating for the tonic production of the gas, thereby maintaining basal cardiovascular function.The effects of melatonin involved NO-dependent mechanisms as the responses were reverted by fetal treatment with the NO clamp.

Mentions:
Following at least 5 days of postoperative recovery, all fetuses were subjected to acute hypoxia experiments, carried out on consecutive days in a randomized order (Fig. 1). Each protocol consisted of a 3-hr period divided into 1.5-hr normoxia, 0.5-hr hypoxia, and 1-hr recovery, during a slow i.v. infusion of either heparinized saline vehicle (0.5% ethanol-99.5% heparinized saline), treatment with a low dose of melatonin (0.05 ± 0.01 μg/kg/min), treatment with a high dose of melatonin (0.5 ± 0.1 μg/kg/min), or treatment with melatonin high during NO blockade with the NO clamp (Fig. 1). The doses of melatonin were calculated retrospectively from the fetal weights (3.4 ± 0.3 kg) obtained at postmortem and were administered to the fetus having been dissolved in 0.5% ethanol-99.5% heparinized saline (80 i.u. heparin/mL in 0.9% NaCl) solution. The doses were chosen to achieve the lowest plasma concentrations compatible with melatonin having the capacity to act as antioxidant [28, 29]. For comparison, the high dose of melatonin used is ca. 32 times lower than that recommended for the prevention of jet lag in humans [30]. The NO clamp is an established technique previously validated in our laboratory that is able to block the production of NO in vivo without affecting basal cardiovascular function [24–26]. In brief, a bolus dose (100 mg/kg dissolved in 2 mL heparinized saline) of L-NAME (NG-nitro-l-arginine methyl ester; Sigma Chemicals, Dorset, UK) was injected via the femoral artery, immediately followed by fetal i.v. infusion with sodium nitroprusside (SNP; Sigma Chemicals; 5.1 ± 2.0 μg/kg/min: mean ± 1 S.D.; dissolved in heparinized saline). The infusion rate of SNP was titrated to avoid any perturbation in basal arterial blood pressure. While fetal treatment with L-NAME alone leads to pronounced systemic vasoconstriction and hypertension, combined treatment of the fetus with both L-NAME and SNP compensates for the tonic production of the gas, maintains basal cardiovascular function, and blocks de novo synthesis of NO during stimulated conditions, such as during acute hypoxia. At the end of the experimental protocol, the effectiveness of NO blockade by the NO clamp and the persistence of L-NAME in the system were tested by withdrawal of the SNP infusion. This unmasked the influence of fetal treatment with L-NAME alone and led to a significant increase in arterial blood pressure, a fall in heart rate, and an increase in femoral vascular resistance (FVR) (data not shown).

Mentions:
Following at least 5 days of postoperative recovery, all fetuses were subjected to acute hypoxia experiments, carried out on consecutive days in a randomized order (Fig. 1). Each protocol consisted of a 3-hr period divided into 1.5-hr normoxia, 0.5-hr hypoxia, and 1-hr recovery, during a slow i.v. infusion of either heparinized saline vehicle (0.5% ethanol-99.5% heparinized saline), treatment with a low dose of melatonin (0.05 ± 0.01 μg/kg/min), treatment with a high dose of melatonin (0.5 ± 0.1 μg/kg/min), or treatment with melatonin high during NO blockade with the NO clamp (Fig. 1). The doses of melatonin were calculated retrospectively from the fetal weights (3.4 ± 0.3 kg) obtained at postmortem and were administered to the fetus having been dissolved in 0.5% ethanol-99.5% heparinized saline (80 i.u. heparin/mL in 0.9% NaCl) solution. The doses were chosen to achieve the lowest plasma concentrations compatible with melatonin having the capacity to act as antioxidant [28, 29]. For comparison, the high dose of melatonin used is ca. 32 times lower than that recommended for the prevention of jet lag in humans [30]. The NO clamp is an established technique previously validated in our laboratory that is able to block the production of NO in vivo without affecting basal cardiovascular function [24–26]. In brief, a bolus dose (100 mg/kg dissolved in 2 mL heparinized saline) of L-NAME (NG-nitro-l-arginine methyl ester; Sigma Chemicals, Dorset, UK) was injected via the femoral artery, immediately followed by fetal i.v. infusion with sodium nitroprusside (SNP; Sigma Chemicals; 5.1 ± 2.0 μg/kg/min: mean ± 1 S.D.; dissolved in heparinized saline). The infusion rate of SNP was titrated to avoid any perturbation in basal arterial blood pressure. While fetal treatment with L-NAME alone leads to pronounced systemic vasoconstriction and hypertension, combined treatment of the fetus with both L-NAME and SNP compensates for the tonic production of the gas, maintains basal cardiovascular function, and blocks de novo synthesis of NO during stimulated conditions, such as during acute hypoxia. At the end of the experimental protocol, the effectiveness of NO blockade by the NO clamp and the persistence of L-NAME in the system were tested by withdrawal of the SNP infusion. This unmasked the influence of fetal treatment with L-NAME alone and led to a significant increase in arterial blood pressure, a fall in heart rate, and an increase in femoral vascular resistance (FVR) (data not shown).

Bottom Line:
Under anesthesia, 6 fetal sheep at 0.85 gestation were instrumented with vascular catheters and a Transonic flow probe around a femoral artery.This technique permits blockade of de novo synthesis of NO while compensating for the tonic production of the gas, thereby maintaining basal cardiovascular function.The effects of melatonin involved NO-dependent mechanisms as the responses were reverted by fetal treatment with the NO clamp.